Pathology - Theses
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ItemMolecular basis of amyloid-β formation: focus on β-secretaseHolsinger, Ramsworth Michael Damian ( 2003)Aβ amyloid deposition is the pathognomonic feature of Alzheimer's disease (AD) and cytotoxic Aβ oligomers are considered to be responsible for neuronal degeneration. Aβ is proteolytically derived from the type 1 amyloid precursor protein (APP) by the sequential action of β- and y-secretases. This thesis focuses on the characterization of the recently identified β-secretase, BACE1 in human brain. This molecule has been propelled to the forefront of AD research due to its potential as a therapeutic target. The work presented here describes the first report of increased BACE1 protein and activity in AD brain. The BACE1 gene was cloned from human brain total RNA and used to transfect mammalian cells. Expressed protein was detected using three BACE1-specific antibodies generated during this candidature. Protein samples were then prepared from human brain and analyzed using BACE1 C-terminal antibody that revealed a significant 2.7-fold increase in BACE1 protein level in AD frontal cortex compared to age-matched and neurological controls. Examination of the C-terminal membrane-bound stub resulting from BACE1 cleavage demonstrated an ~2-fold increase in β-CTF levels confirming elevated enzyme activity. A more detailed analysis aimed at elucidating the mechanism by which BACE1 protein levels are increased in AD brain showed that this did not occur at the level of the message since BACE1 mRNA levels did not differ between control and AD groups. Analysis of lipid raft fractions prepared from AD and control brains demonstrated the presence of increased levels of amyloid generating machinery as well as amyloidogenic Aβ in rafts. Furthermore, it was also discovered by Western blotting that multiple BACE1 immunoreactive species were present in human brain and that this immunoreactivity was increased in more dense fractions that also contained lipid rafts. The second area of research describes the generation of recombinant BACE1 protein in a mammalian cell expression system. Cloning and characterization of the putative ectodomain construct revealed that a six amino acid sequence previously believed to be part of the ectodomain was sufficient to retain BACE1 within the cell, associated with the membrane as determined by biochemical, enzymatic and microscopic techniques. Removal of these six amino acids resulted in a secreted product that was purified from culture medium by anion chromatography coupled to FPLC. Using techniques developed in this section, lipid raft fractions were examined for BACE1 enzymatic activity and it was discovered that elevated levels of activity were associated with dense fractions. These results add support to those from Western blotting described above that showed increased BACE1 immunoreactivity coupled with that of other amyloidogenic proteins segregating in these dense fractions suggesting possible compartments where Aβ generation may occur. Work presented in the final section describes the first report of BACE1 enzyme activity measured in cerebrospinal fluid. Using a quenched fluorescence enzyme assay BACE1 activity was found to be elevated 3-fold in post-mortem AD CSF compared to control. This finding suggests the possibility of using BACE1 as a biological tool in the diagnosis of Alzheimer's disease. The work presented in this thesis describes the analysis of the β-secretase enzyme BACE1 in human brain. We have shown that BACE1 protein and activity are increased in AD brain and have identified probable compartments where this may occur. We have also shown that BACE1 activity is increased in AD CSF identifying this enzyme as a potential diagnostic marker of the disease.